SAMHD1 is a deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase. It is a host cell restriction factor that plays critical roles in the defense against human immunodeficiency virus 1 (HIV-1) infection. SAMHD1 lowers the cellular concentrations of dNTP such that effective reverse transcription is prohibited. Interestingly, SAMHD1 also possesses nuclease activity, shown to be essential for its HIV-1 restriction. Understanding the molecular and structural basis of these two important biological activities and their regulation will provide important insight into mechanisms of HIV restriction. To this end, we propose the following Aims: In Aim 1, we will determine the structural basis and molecular mechanisms of SAMHD1 dNTPase activity by nucleoside triphosphates. In Aim 2, the structure-function relationship of the intra- and inter- domain interactions will be investigated. In Aim 3, e will characterize the nuclease activity of SAMHD1 and its structural determinants. Results from this work will provide avenues for designing new therapeutic approaches to fight AIDS.